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Genetically Engineered Fusion Proteins for Treatment of Cancer

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Genetically Engineered Fusion Proteins for Treatment of Cancer CANCER GENOMICS & PROTEOMICS 9: 357-372 (2012) Review Genetically Engineered Fusion Proteins for Treatment of Cancer ULRICH H. WEIDLE, BRITTA SCHNEIDER, GUY GEORGES and ULRICH BRINKMANN Roche Pharma Research and Early Development (pRED), Roche Diagnostics GmbH, Penzberg, Germany Abstract. In this review, we summarize approaches to treat generated by combining entities derived from the ECDs of cancer with genetically engineered fusion proteins. Such two, or theoretically more, plasma membrane-associated proteins can act as decoy receptors for several ligands or as receptors. The fusion proteins, as outlined, can bind several recruiters of immune effector cells to tumor. Examples of ligands dedicated to one or several receptors inhibiting, interference with growth factor-mediated tumor growth and processes such as tumor-associated angiogenesis or human tumor-related angiogenesis with fusion proteins consisting of epidermal growth factor receptor (HER)-based signaling. the extracellular domains, and in some cases also of entities Another approach is the recruitment of immune effector cells of one or several receptors and the Fc part of human IgG1, to the tumor with genetically engineered fusion proteins. We are discussed. In addition, we present strategies for discuss fusion proteins of tumor-related antibodies and recruitment of immune effector cells to tumor with fusion cytokines or MHC-class I peptide complexes, T-cell receptor proteins. This can be achieved with fusion proteins consisting (TCR) cytokine fusion proteins, and fusions proteins of a of a tumor-related antibody and a cytokine or major T-cell-recruiting antibody and a tumor-cell receptor ligand or histocompatibilty complex class-I-peptide complexes, by a defined TCR. T-cell receptor cytokine fusion proteins or by combination of a T-cell-recruiting antibody with a tumor-related ligand or a Technologies for Generation of Genetically defined T-cell receptor. Engineered Fusion Proteins One of the aspects of biologics-based cancer therapy is the Genetically engineered fusion proteins are composed of inhibition of angiogenesis and of tumor-promoting effects of domains of antibodies (predominantly IgG), and of growth factors. Another approach is based on the recruitment additional protein entities that mediate functionality. The of immune effector cells to the tumor in order to initiate an entities that are derived from antibodies can either contain antitumor immune response. These approaches are based on binding regions or constant antibody regions. the identification of the hallmarks of cancer (1-3). Using the Binding region modules are applied for targeting first approach, several recombinant receptor decoys are under approaches, i.e. to direct a fused ‘effector’ payload pre-clinical and clinical investigation. They are based on specifically to those cells that shall be addressed. These genetic fusion of the extracellular domain (ECD) of a binding entities are composed, in most cases, of variable receptor and the Fc-part of human immunoglobulin IgG1, regions (Fv) of antibodies which specifically bind the desired which improves the pharmacokinetic properties of the fusion antigen. Fv fragments are heterodimers of variable heavy protein. Chimeric decoy receptor fusion proteins can be chain (VH) and variable light chain (VL) domains. They are the smallest modules that retain a complete structure of the binding region of antibodies. Their small size (approx. This article is freely accessible online. 25 kDa) enables the generation of small fusion proteins which may penetrate tissues and tumors quite rapidly. Such Correspondence to: Ulrich H. Weidle, Roche Pharma Research and molecules may be particularly useful in applications that Early Development (pRED), Roche Diagnostics GmbH, Im require rapid tissue/tumor penetration, e.g. for targeted cancer Nonnenwald 2, D-82372 Penzberg, Germany. E-mail: therapy. The complete removal of the constant regions of [email protected] antibodies not only reduces the size of fusion proteins, but it Key Words: Angiogenesis, antibody cytokine fusion proteins, also restricts the functionality of antibody modules to chimeric extracellular receptor domains, decoy receptor, HER ‘targeting-only’. Removal of the constant domains eliminates signaling; Fc-based fusion proteins, immune effector cell any (Fc-receptor mediated) effector functionality of the parent recruitment, treatment resistance, review. antibody, such as complement-dependent cytotoxicity (CDC) 1109-6535/2012 $2.00+.40 357 CANCER GENOMICS & PROTEOMICS 9: 357-372 (2012) or antibody-dependent cellular cytotoxicity (ADCC). the modulation of size and the pharmacokinetics of the Application of Fvs without attached constant domains therapeutic fusion proteins. The Fc-fragment of antibodies generates technical challenges, in particular stability issues: (IgG) starts with an N-terminal at the flexible hinge region of while fragment antigen binding (Fabs), [heterodimers antibodies, thereafter followed by CH2 and CH3 domains. composed of VH first constant domain of the heavy chain These entities form stable homodimers, covalently linked by (CH1) and VL (CL)] are by themselves quite stable interchain disulfide bonds between cysteine residues in their heterodimers, VH-VL heterodimers are instable. The CL and hinge regions. The fusion partners of Fc-containing therapeutic CH1 domains contribute significantly to the formation of the proteins are connected to each other in a combinatorial heterodimeric Fabs, and their interactions (including an manner. In most cases, the fusion to Fc is made at the hinge interchain disulfide at the C-terminal end of CH1 and CL) region, followed thereafter by CH1 and CH2. Hinge regions stabilize Fab fragments. Once these domains are removed, are flexible by nature, thus fusion at this position minimizes VH and VL have only very limited capability to form or prevents undesired interactions and steric hindrance. This functional Fv heterodimers. Furthermore, VH-VL interfaces combinatorial composition retains the functionality of the Fc are too weak to stably retain heterodimeric Fvs without CH1 region, as well as that of the additional entity, as will be shown and CL attached (4, 5). One method that has frequently and in this review. Constant regions bind Fc receptors, including successfully been applied to overcome the problem of instable FcRn (12, 13). Binding to FcRn, which enables antibody VH-VL heterodimers is the covalent connection of VH and recycling/export from cells, is the key contributor to the long VL by a flexible peptide linker. These linkers are frequently serum half-life of antibodies. This favorable pharmacokinetic composed of glycine and serine, containing stretches of 15- property can also be transferred to fusion proteins that contain 20 amino acids. The tethering between VH and VL generates Fc domains as FcRn-binding modules. A further advantage of single-chain Fv molecules (6, 7), which are more stable having Fc regions as part of recombinant fusion proteins is compared to VH-VL heterodimers by themselves. Single- that they can be applied to facilitate production and chain (sc)Fvs are widely applied as recombinant binding downstream processing. For this reason, Fc fusions can be modules and components of fusion proteins. expressed and purified by procedures that are similar to those Additional technologies have been applied to further applied for the production of therapeutic antibodies. This stabilize Fv fragments for therapeutic applications. For some includes expression in mammalian cells, secretion into culture variable regions, linkage of VH and VL by flexible peptide supernatants and subsequent affinity-based (ProteinA) linkers only partially restores its stability. Such scFvs still purification steps. Examples of genetically engineered fusion have a tendency to dissociate and in consequence have a proteins that contain constant regions of antibodies are ‘ligand propensity to aggregate. These problems can be overcome by trap’ molecules such as those containing extracellular domains increasing the stability of the VH-VL interface by mutations of fibroblast growth factor (FGF), epidermal growth factor and/or by introduction of covalent disulfide connections receptor (EGFR), human epidermal growth factor receptor-2 between the VH and VL domains (8-10). The fusion partners (HER2), and of vascular endothelial growth factor (VEGF) of Fv-containing proteins are connected to each other in a receptors (Figure 2, A-F). modular manner. In most cases, flexible linker or connector sequences are set between the Fv module and the additional VEGF Trap entity to prevent undesired interactions (including steric hindrance) between the fusion partners. This modular The members of VEGF family are involved in processes such composition retains the binding specificity of the Fv region as angiogenesis, lymphangiogenesis, vasculogenesis, and are as well as the functionality of the targeted-effector entity. also modulators of host innate and adoptive immunity (14). Engineered Fv fusion proteins can be efficiently produced in Clinical studies have validated VEGF as a target for treatment bacteria such as Escherichia coli, either as soluble proteins of cancer and vascular diseases affecting the eye. These studies or as inclusion bodies, followed by re-folding to obtain fully have led to the approval of drugs targeting the VEGF pathway, active proteins (11). Examples for genetically engineered such as an antibodies directed against VEGF-A (Avastin), and fusion
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